Common micro- and nanoscale subcellular compartments are formed from either lipids or proteins and include mitochondria, lysosomes, peroxisomes, carboxysomes and other metabolosomes, as well as multi-enzyme complexes. Compartments increase the overall activity and specificity of the encapsulated enzyme pathways by maintaining a high local concentration of enzymes and substrates, promoting substrate channeling and protecting their content from damage, as well as by segregating potentially damaging reactions from the cytosol. Spatial confinement is also an important aspect for chaperone-assisted folding of linear polypeptides into active tertiary and quaternary conformations, as well as for preventing proteins from aggregating under cellular stress conditions. A better understanding of the effects of spatial confinement on protein function will not only enhance the fundamental knowledge of cellular organization and metabolism but also increase the ability to translate biochemical pathways into a variety of noncellular applications, ranging from diagnostics and drug delivery to the production of high-value chemicals and smart materials. Over the past few decades, artificial enzymatic particles have been created using compartmentalization by virus-like protein particles, liposomes or polymersomes and chemical crosslinking. However, severe obstacles to a broader application remain, including low encapsulation yield of large proteins because of steric hindrance, insufficient access of substrates to the encapsulated enzymes, aggregation of vesicle shells and limited control over the spatial arrangement of proteins within the compartments.